Nontypeable Haemophilus influenzae (NTHI) is an important human pathogen in several settings. It is especially important as a cause of otitis media in children. Otitis media represents as enormous national health problem both from the point of view of human suffering and of cost. Approximately 80% of children will have had at least one episode of otitis media by age 3. Otitis media is the most common reason for visits to pediatricians. Recurrent otitis media is painful; it is associated with delay in speech and language development in infants and children; and it is associated with learning problems as children reach school age. The annual direct cost of medical care for otitis media nationally is estimated to be $2 billion. A vaccine to prevent otitis media would go a long way toward relieving human suffering and reducing this financial burden on the national health care system. That is one of the goals of the work proposed. Work in the previous funding period has focused on studies of the antigenic structure of two outer membrane proteins (OMPs) of NTHI. This work has led to the identification of the P6 OMP as a promising antigen to be used in a vaccine. The P2 molecule has been identified as an important antigen in the immune response to NTHI. Our hypothesis is that P2 expresses immunodominant epitopes to which otitis prone children preferentially make strain specific antibodies leaving them susceptible to recurrent episodes of infection with heterologous strains. We further hypothesize that antibodies to P6 and/or to conserved surface-exposed regions of P2 will be protective from infection by many strains. The goals of this proposal are to test these hypotheses. To understand the role of the P2 OMP in interacting with tee host, monoclonal antibodies to conserved surface-exposed loops on the P2 molecule will be developed and characterized. The immune response in otitis-prone children to the potentially immunodorninant regions of the homologous P2 molecule will be characterized. To further assess the vaccine potential of the P6 OMP, potentially protective B cell epitopes and T cell epitopes will be identified. A novel molecule to effectively present protective epitopes to the host will be constructed. The results of the proposed studies will contribute important information on the antigenic structure of two important molecules on the surface of NTHI and will help elucidate the importance of these molecules in the human immune response to NTHI in otitis media. This information will be critical in the successful development of a vaccine to prevent otitis media and other infections caused by NTHI.